U.S. patent application number 17/441588 was filed with the patent office on 2022-06-16 for virus-like particles and uses thereof.
This patent application is currently assigned to GEORGIA STATE UNIVERSITY RESEARCH FOUNDATION, INC.. The applicant listed for this patent is GEORGIA STATE UNIVERSITY RESEARCH FOUNDATION, INC.. Invention is credited to Christopher F. BASLER, JoAnn M. TUFARIELLO, Lin WANG.
Application Number | 20220184200 17/441588 |
Document ID | / |
Family ID | 1000006227482 |
Filed Date | 2022-06-16 |
United States Patent
Application |
20220184200 |
Kind Code |
A1 |
BASLER; Christopher F. ; et
al. |
June 16, 2022 |
VIRUS-LIKE PARTICLES AND USES THEREOF
Abstract
Provided herein are virus-like particles and their uses for
stimulating anti-pathogenic and anti-cancer immune responses.
Inventors: |
BASLER; Christopher F.;
(Atlanta, GA) ; TUFARIELLO; JoAnn M.; (Atlanta,
GA) ; WANG; Lin; (Atlanta, GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GEORGIA STATE UNIVERSITY RESEARCH FOUNDATION, INC. |
Atlanta |
GA |
US |
|
|
Assignee: |
GEORGIA STATE UNIVERSITY RESEARCH
FOUNDATION, INC.
Atlanta
GA
|
Family ID: |
1000006227482 |
Appl. No.: |
17/441588 |
Filed: |
March 20, 2020 |
PCT Filed: |
March 20, 2020 |
PCT NO: |
PCT/US2020/023911 |
371 Date: |
September 21, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62821744 |
Mar 21, 2019 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/572 20130101;
A61K 2039/70 20130101; C12N 7/00 20130101; A61K 2039/55516
20130101; A61P 31/14 20180101; A61K 2039/575 20130101; A61K
2039/5258 20130101; A61K 2039/545 20130101; A61K 39/04 20130101;
A61P 31/06 20180101; A61K 39/12 20130101 |
International
Class: |
A61K 39/12 20060101
A61K039/12; A61K 39/04 20060101 A61K039/04; A61P 31/06 20060101
A61P031/06; A61P 31/14 20060101 A61P031/14; C12N 7/00 20060101
C12N007/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
[0002] This invention was made with government support under
AI109664, AI109945, AI114654, AI127009, AI125453, AI109965, and
AI127835 awarded by the National Institutes of Health and under
HDTRA1-16-1-0033 and HDTRA1-17-1-0005 awarded by the Department of
Defense. The government has certain rights in the invention.
Claims
1. A virus-like particle (VLP) comprising: a. a surface protein; b.
a matrix protein; c. a polypeptide that enhances an immune
response, wherein the polypeptide is a polypeptide adjuvant and
wherein the polypeptide is linked to the surface protein, the
matrix protein, or an intra-VLP protein.
2. The VLP of claim 1, wherein the surface protein induces a B-cell
mediated immune response.
3. The VLP of claim 1 or 2, wherein the intra-VLP protein or a
fragment thereof induces a T cell-mediated immune response.
4. The VLP of any one of claims 1-3, wherein the polypeptide
adjuvant enhances a B-cell mediated immune response, a T
cell-mediated immune response, or both a B-cell mediated immune
response and a T cell-mediated immune response.
5. The VLP of any one of claims 1-4, wherein the polypeptide
adjuvant comprises one or more signaling domains.
6. The VLP of claim 5, wherein the one or more signaling domains
comprise caspase activation and recruitment domains (CARDs).
7. The VLP of claim 5 or 6, wherein the polypeptide induces a Type
I interferon immune response.
8. The VLP of any one of claims 1-7, wherein the immune response is
an anti-pathogenic immune response.
9. The VLP of claim 7, wherein the anti-pathogenic immune response
is an antiviral immune response, an antibacterial immune response,
an antifungal immune response, or an antiparasitic immune
response.
10. The VLP of any one of claims 1-7, wherein the immune response
is an anticancer immune response
11. The VLP of any one of claims 1-9, wherein the surface protein
comprises a bacterial protein.
12. The VLP of any one of claims 1-9, wherein the matrix protein or
the intra-VLP protein comprises a bacterial protein.
13. The VLP of claim 11 or 12, wherein the bacterial protein is a
mycobacterial protein.
14. The VLP of any one of claim 1-7 or 10, wherein the surface
protein comprises a cancer antigen.
15. The VLP of any one of claims 1-14, wherein the surface protein
comprises a viral surface protein.
16. The VLP of any one of claims 1-15, wherein the matrix protein
comprises a viral matrix protein.
17. The VLP of any one of claims 1-16, wherein the intra-VLP
protein or a fragment thereof comprises a viral nucleoprotein (NP)
or a fragment thereof.
18. The VLP of claim 17, wherein the viral NP fragment is a
C-terminal fragment of a viral NP.
19. The VLP of claim 17 or 18, wherein the viral surface protein,
the viral matrix protein and the viral NP or a fragment thereof are
from the same virus.
20. The VLP of claim 19, wherein the virus is selected from the
group consisting of a filovirus, an arenavirus, a paramyxovirus, a
pneumovirus, and an influenza virus.
21. The VLP of claim 17 or 18, wherein the viral surface protein,
the viral matrix protein and the viral NP or a fragment thereof are
from different strains of the same virus or different viruses.
22. The VLP of claim 19, wherein the different strains or different
viruses are selected from the group consisting of a filovirus, an
arenavirus, a paramyxovirus, a pneumovirus, and an influenza
virus.
23. The VLP of any one of claims 5-22, wherein the intra-VLP
protein or a fragment thereof is linked to a polypeptide comprising
two signaling domains.
24. The VLP of any one of claims 5-23, wherein at least one
signaling domain is a CARD domain.
25. The VLP of claim 24, wherein the at least one signaling domain
is a CARD domain from a RIG-I-like receptor.
26. The VLP of claim 25, wherein the RIG-I-like receptor is
retinoic acid-inducible gene-I (RIG-I).
27. The VLP of claim 25, wherein the RIG-I-like receptor is
Melanoma Differentiation-Associated protein 5 (MDA5).
28. The VLP of any one of claims 16-27, wherein the viral matrix
protein is an Ebola virus VP40 matrix protein.
29. The VLP of claim 28, wherein the viral surface protein is an
Ebola virus glycoprotein (GP).
30. The VLP of claim 29, wherein the viral NP is an Ebola virus NP
or a fragment thereof.
31. The VLP of claim 21, wherein the viral surface protein and the
viral NP or a fragment thereof are from a different strain or a
different virus.
32. The VLP of any one of claims 15-27, wherein the viral surface
protein is a Lassa virus GPC.
33. The VLP of claim 32, wherein the viral matrix protein is a
Lassa virus matrix protein Z.
34. The VLP of claim 32 or 33, wherein the viral nucleoprotein is a
Lassa virus NP or a fragment thereof.
35. The VLP of any one of claims 15-27, wherein the viral surface
protein is an influenza virus glycoprotein.
36. The VLP of claim 35, wherein the influenza virus glycoprotein
is an influenza hemagglutinin (HA).
37. The VLP of claim 35 or 36, wherein the viral matrix protein is
an influenza M1 protein.
38. The VLP of claim 37, wherein the viral nucleoprotein is an
influenza virus NP or a fragment thereof.
39. The VLP of any one of claims 15-27, wherein the viral surface
protein is a henipavirus glycoprotein.
40. The VLP of claim 39, wherein the henipavirus glycoprotein is a
Nipah virus F protein or a Nipah virus G protein.
41. The VLP of claim 39 or 40, wherein the viral matrix protein is
a Nipah virus matrix (M) protein.
42. The VLP of claim 41, wherein the viral nucleoprotein is Nipah
virus NP or a fragment thereof.
43. An isolated host cell that expresses the VLP of any one of
claims 1-42.
44. The isolated host cell of claim 43, wherein the host cell is a
mammalian cell.
45. The isolated host cell of claim 44, wherein the host cell is an
insect cell.
46. An immunogenic composition comprising the VLP of any one of
claims 1-42 and a pharmaceutically acceptable carrier.
47. A method of making the immunogenic composition of claim 46
comprising: (a) expressing in a host cell at least one VLP of any
one of claims 1-42; (b) growing the host cell under conditions
which allow the formation of VLPs; (c) purifying the VLPs, and (d)
preparing the immunogenic composition with the purified VLPs.
48. The method of claim 47, wherein the host cell is transfected or
infected with one or more recombinant constructs encoding (a) the
surface protein; (b) the matrix protein, (c) the intra-VLP protein
or a fragment thereof; and (d) a polypeptide that enhances an
immune response, wherein the polypeptide is linked to the surface
protein, the matrix protein or the intra-VLP protein or a fragment
thereof.
49. An immunogenic composition comprising at least one VLP produced
by the method of claim 47 or 48.
50. A method of stimulating an immune response in a subject
comprising administering to the subject an effective amount of the
immunogenic composition of claim 49.
51. The method of claim 50, wherein the immunogenic composition is
administered to the subject as a single dose.
52. The method of claim 50 or 51, wherein the immunogenic
composition enhances an immune response in the subject.
53. The method of any one of claims 50-52, wherein the immune
response is an immune response against a pathogen in the
subject.
54. The method of any one of claims 50-52, wherein the immune
response is an anticancer immune response.
55. The method of claim 53, wherein the pathogen is a virus, a
bacterium, a fungus, or a parasite.
56. The method of claim 55, wherein the virus is selected from the
group consisting of a filovirus, an arenavirus, a henipavirus, a
pneumovirus, and an influenza virus.
57. The method of claim 56, wherein the virus is selected from the
group consisting of Ebola virus, Nipah virus, Lassa virus and
influenza virus.
58. The method of claim 53, wherein the pathogen is a
bacterium.
59. The method of claim 58, wherein the bacterium is a
mycobacterium.
Description
PRIOR RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/821,744 filed on Mar. 21, 2019, which is hereby
incorporated by reference in its entirety.
BACKGROUND
[0003] Virus-like particles (VLPs) are multi-protein structures
that mimic the organization and conformation of authentic native
viruses but lack the viral genome, potentially yielding safer and
cheaper vaccine candidates. Since VLPs resemble a virus, they can
present antigens in their native state to elicit both B and T cell
responses. However, despite some advantages, the use of VLPs as
vaccines has encountered several difficulties, including
inconsistent manufacture of VLPs and relatively low immunogenicity,
as compared to live viruses.
SUMMARY
[0004] The present disclosure is based, at least in part, on the
discovery of VLPs with enhanced immunogenicity. These VLPs contain
a polypeptide adjuvant, i.e., a self-adjuvant, and can be used to
stimulate an immune response in a subject, for example, an
anti-pathogenic response or an anti-cancer response. The VLPs
comprise a surface protein, a matrix protein, a polypeptide that
enhances an immune response, and optionally, an intra-VLP protein
or a fragment thereof, wherein the polypeptide is a polypeptide
adjuvant and wherein the polypeptide is linked to the surface
protein, the matrix protein or the intra-VLP protein.
[0005] In some embodiments, the surface protein induces a B-cell
mediated immune response. In some embodiments, the surface protein
is a targeting protein. In some embodiments, the intra-VLP protein
or a fragment thereof induces a T cell-mediated immune
response.
[0006] The polypeptide adjuvant enhances a B-cell mediated immune
response, a T cell-mediated immune response, or both a B-cell
mediated immune response and a T cell-mediated immune response. In
some embodiments, the polypeptide adjuvant comprises one or more
signaling domains, such as caspase activation and recruitment
domains (CARDs). In some embodiments, the polypeptide adjuvant
induces a Type I interferon immune response. In some embodiments,
the polypeptide adjuvant comprises a protein or fragment thereof
that induces cytokine or chemokine responses, with or without
inducing a Type I interferon immune response.
[0007] The immune response is optionally an anti-pathogenic immune
response. For example, the anti-pathogenic immune response can be
an antiviral immune response, an antibacterial immune response, and
anti-fungal immune response, or an anti-parasitic immune response.
In some embodiments, the immune response is an anticancer immune
response
[0008] The surface protein optionally comprises a cancer antigen or
a bacterial protein. For example, the bacterial protein can be a
mycobacterial protein. The bacterial protein can be a bacterial
surface protein or a secreted protein. By way of further example,
the surface protein can comprise ESAT6 (an exemplary mycobacterial
protein), Ag85, or a fragment thereof. Example of VLPs that
enhances an anti-bacterial immune response include (a) Ebola virus
GP, Ebola virus VP40 and bacterial antigen fused to 2CARD-Ebola
NPct and (b) Ebola virus GP, bacterial antigen fused to Ebola virus
VP40 and 2CARD-bacterial antigen-Ebola virus NPct.
[0009] In some embodiments, the surface protein comprises a viral
surface protein, the matrix protein is a viral matrix protein,
and/or the intra-VLP protein or a fragment thereof is a viral
nucleoprotein (NP) or a fragment thereof. By way of example, the
viral NP fragment can be a C-terminal fragment of a viral NP (e.g.,
a fragment of Ebola virus NP having the amino acid sequence of SEQ
ID NO:1).
[0010] Optionally, the viral surface protein, the viral matrix
protein and the viral NP or a fragment thereof are from the same
virus. The virus can be selected from the group consisting of a
filovirus, an arenavirus, a paramyxovirus (e.g., a henipavirus),
pneumovirus (e.g., respiratory syncytial virus), and an influenza
virus.
[0011] In some embodiments, the viral surface protein, the viral
matrix protein and the viral NP or a fragment thereof are from
different viruses. Thus, the viral surface protein, the matrix
protein, and the viral NP can be selected from the group consisting
of a filovirus, an arenavirus, a paramyxovirus (e.g., a
henipavirus), a pneumovirus (e.g., respiratory syncytial virus),
and an influenza virus.
[0012] The intra-VLP protein or a fragment thereof is optionally
linked to a polypeptide comprising two signaling domains (e.g.,
CARDs). In some embodiments, at least one CARD domain is a CARD
domain from a RIG-I-like receptor (e.g., 2 CARD domains from the
N-terminus of RIG-I). For example, the RIG-I-like receptor can be
retinoic acid-inducible gene-I (RIG-I), Melanoma
Differentiation-Associated Protein 5 (MDA5), or laboratory of
genetics and physiology 2 (LGP2) (the product of the DHX58 gene).
Similarly, a different interferon inducing domain or protein can be
used. Examples include the proteins or domains from TIR Domain
Containing Adaptor Inducing Interferon-Beta (TRIF) or STING
(Stimulator Of Interferon Genes Protein).
[0013] In some embodiments, the viral matrix protein is an Ebola
virus VP40 matrix protein, the viral surface protein is an Ebola
virus glycoprotein (GP) and the viral NP is an Ebola virus NP or a
fragment thereof. Optionally, the surface protein and/or the viral
NP or a fragment thereof are from different members of the same
virus family (e.g., different strains of the filovirus family) or
from different viruses (e.g., a filovirus and a non-filovirus
virus).
[0014] In some embodiments, the viral surface protein is a Lassa
virus GPC, the viral matrix protein is a Lassa virus matrix protein
Z and/or the viral nucleoprotein is a Lassa virus NP or a fragment
thereof.
[0015] In some embodiments, the viral surface protein is an
influenza virus glycoprotein. In some embodiments, the influenza
virus glycoprotein is an influenza virus hemagglutinin (HA), the
viral matrix protein is an influenza virus M1 protein and/or the
viral nucleoprotein is an influenza virus NP or a fragment
thereof.
[0016] In some embodiments, the viral surface protein(s) is (are) a
henipavirus glycoprotein(s), such as a Nipah virus F protein and/or
Nipah virus G protein; the viral matrix protein is a Nipah virus
matrix (M) protein; and the viral nucleoprotein is Nipah virus N or
a fragment thereof.
[0017] In some embodiments, the viral surface protein is a
respiratory syncytial virus (RSV) glycoprotein, such as a RSV F
protein and/or RSV virus G protein; the viral matrix protein is a
RSV matrix (M) protein; and the viral nucleoprotein is RSV N or a
fragment thereof.
[0018] Also provided is an isolated host cell that expresses any of
the VLPs described herein. In some embodiments, the host cell is a
mammalian cell or an insect cell.
[0019] Further provided is an immunogenic composition comprising
any of the VLPs described herein and a pharmaceutically acceptable
carrier.
[0020] Also provided are methods of making any of the VLPs or
immunogenic compositions described herein. The methods include the
steps of (a) expressing in a host cell at least one of the VLPs
described herein; (b) growing the host cell under conditions that
allow the formation of VLPs; and (c) purifying the VLPs.
Optionally, the method further comprises preparing the immunogenic
composition with the purified VLPs by adding a pharmaceutically
acceptable carrier.
[0021] In some methods of making VLPs or immunogenic compositions
comprising a VLP, the host cell is transfected or infected with one
or more recombinant constructs encoding (a) the surface protein;
(b) the matrix protein, (c) optionally the intra-VLP protein or a
fragment thereof; and (d) a polypeptide that enhances an immune
response, wherein the polypeptide is linked to the surface protein,
the matrix protein or the intra-VLP protein or a fragment
thereof.
[0022] Further provided is an immunogenic composition comprising at
least one VLP produced by any of the methods provided herein.
[0023] Also provided is a method of stimulating an immune response
in a subject comprising administering to the subject an effective
amount of any of the immunogenic compositions provided herein. The
immunogenic composition can be administered to the subject as a
single dose or as multiple doses (e.g., initial immunization plus
one or more boosts). In some embodiments, the immunogenic
composition enhances an immune response in the subject. The immune
response can be an anticancer immune response or an immune response
against a pathogen. The pathogen can be, for example, a virus, a
bacterium, a fungus, or a parasite. The virus, for example, can be
selected from the group consisting of a filovirus, an arenavirus, a
paramyxovirus (including a henipavirus), a pneumovirus, and an
influenza virus. In some embodiments, the virus is selected from
the group consisting of Ebola virus, Nipah virus, Lassa virus and
influenza virus. In some embodiments, the pathogen is a bacterium,
such as a mycobacterium.
BRIEF DESCRIPTION OF THE FIGURES
[0024] The present application includes the following figures. The
figures are intended to illustrate certain embodiments and/or
features of the compositions and methods, and to supplement any
description(s) of the compositions and methods. The figures do not
limit the scope of the compositions and methods, unless the written
description expressly indicates that such is the case.
[0025] FIG. 1A is a diagram illustrating exemplary constructs used
to generate various Ebola VLPs (eVLPs). eVLPs were generated by
co-expressing a Flag-tagged Ebola virus VP40 matrix protein and
Ebola virus glycoprotein (GP). These were co-transfected with empty
vector or plasmids that produced hemagglutinin epitope (HA)-tagged
full-length Ebola virus nucleoprotein (NP.sub.FL), NP C-terminal
domain (NP.sub.CT), or a region of retinoic acid-inducible gene-I
(RIG-I) protein encompassing the two RIG-I caspase activation and
recruitment domains (2CARD) fused to NP.sub.CT via a Ser-Gly linker
(2CARD NP.sub.CT).
[0026] FIG. 1B shows transmission electron microscopic (EM) images
of eVLPs produced with the 2CARD-NP.sub.CT construct. The left
panel shows the filamentous and curved forms characteristic of
Ebola virus (EBOV) virions and eVLPs. The right panel shows
immunogold labeling of GP on the eVLP membranes with EM images at
higher magnification.
[0027] FIG. 1C shows Western blots of purified eVLPs, with blotting
for HA-NP constructs, Flag-VP40 and GP. The left panel shows a blot
of eVLPs after purification. The right panel shows a blot of
purified eVLPs after trypsin-treatment to remove extra-VLP
protein.
[0028] FIG. 2 shows that the 2CARD-NP.sub.CT construct, like a
2CARD without fusion to NP.sub.CT (2CARD), induces an interferon
(IFN) response. Constructs co-transfected into cells to assess
activation of the IFN.beta. promoter are shown at the top. A
full-length RIG-I construct (RIG-I), in which the 2CARD domain is
fused to the regulatory helicase domain was included as a control
and illustrates that full-length RIG-I does not induce an IFN
response in the absence of an activator. 2CARD is the RIG-I
signaling domain separated from the regulatory domain, resulting in
constitutive signaling activity and expression of the reporter
gene. 2CARD-NP.sub.CT is the fusion of 2CARD to the NP C-terminal
domain (CTD). Empty vector was used as a negative control.
IFN.beta.-luciferase contains the IFN.beta. promoter upstream of
firefly luciferase. Renilla luciferase refers to a reporter plasmid
in which Renilla luciferase is constitutively expressed. The lower
panel shows relative induction of the IFN.beta. promoter by the
2CARD and 2CARD-NP.sub.CT constructs. Dual luciferase assays were
performed 24 hrs post-transfection. Firefly luciferase activity was
normalized to Renilla luciferase activity. The data are reported as
fold-change of firefly luciferase relative to the empty vector
control.
[0029] FIG. 3 shows quantitative RT-PCR measurement of expression
of IFN.beta., IFN-inducible RIG-I and ISG15 and TNF-.alpha. mRNA
levels following addition of eVLPs containing 2CARD-NP.sub.CT to
cells after three hours (left column for each VLP), six hours
(middle column for each VLP) and twelve hours (right column for
each VLP). 2CARD-eVLPS stimulated responses comparable to
transfected polyI:C, an activator or RIG-I. The responses required
the presence of the 2CARD domain, as eVLPs that contained either
NP.sub.FL or NP.sub.CT without 2CARD did not activate the response.
Values were normalized to .beta. actin mRNA levels and reported as
fold-change relative to VLPs produced by VP40 alone.
[0030] FIG. 4. demonstrates that induction of IFN.beta.,
IFN-inducible RIG-I, ISG56 and IRF-7 as well as cytokine
TNF-.alpha. mRNA levels depends on the presence of MAVS, a
signaling molecule downstream of RIG-I that is required for
RIG-I-dependent induction of IFN responses. eVLPs with full-length
NP (NP.sub.FL VLP), with NP.sub.CT or with 2CARD-NP.sub.CT were
used to infect A549 cells or A549 cells in which MAVS had been
deleted. For these experiments, infection with Sendai virus (SeV)
served as a positive control activator of IFN signaling. Only the
2CARD-NP.sub.CT VLP and SeV induced expression of the measured
genes at 4, 8 and 16 hours post-infection in the A549 cells.
Induction was abrogated in the MAVS knockout cells, consistent with
a model whereby the 2CARD-NP.sub.CT VLPs induce IFN responses and
TNF.alpha. production via the canonical RIG-I pathway.
[0031] FIG. 5 shows sustained anti-GP responses of mice at 2, 4, 6
and 8 weeks after a single immunization with 2CARD-NP.sub.CT eVLPs.
Mice were immunized with the indicated eVLP preparations, bled at
the indicated time points and antibody titers assessed by ELISA
using recombinant GP as an antigen. PBS injection served as a
negative control immunization. 10 .mu.g of eVLP was given to all
mice by the intraperitoneal (IP) route, except for the 2CARD-eVLP
high dose group which received 25 .mu.g by the IP route.
[0032] FIG. 6A provides schematics of the constructs used to make
VLPs containing 2CARD and ESAT6, an exemplary Mycobacterial
tuberculosis antigen.
[0033] FIG. 6B and FIG. 6C show IFN responses induced by VLPs
containing both 2CARD and the ESAT6 Mtb antigen. HEK293T cells were
transfected with plasmids encoding the following proteins:
GP+eVP40; GP+eVP40+2CARD-NP.sub.CT; GP+ESAT6-eVP40;
GP+2CARD-ESAT6-eVP40; 2CARD-ESAT6-eVP40. After harvesting and
purifying the VLPs, the VLPs were added to HEK293T cells. As a
control, the HEK293T cells were mock treated. Activation of innate
immune responses were determined by measuring levels of mRNAs to
interferon .beta.(IFN.beta.) and the IFN.beta.-induced gene RIG-I.
These mRNA levels were normalized to .beta.-actin mRNA levels and
reported as relative copies of the IFN.beta. (FIG. 6B) or RIG-I
mRNAs (FIG. 6C).
[0034] FIG. 7 shows that eVLPs produced with a 2CARD-ESAT6-VP40
construct induce an IFN response in a MAVS-dependent manner.
Wildtype A549 or MAVS-knockout A549 cells were mock infected or
infected with eVLPs of the following types: GP+eVP40,
GP+2CARD-NPct, GP+ESAT6-eVP40, GP+2CARD-ESAT6-VP40. Infection was
performed by adding eVLPs to the cells. Twelve hours
post-Infection, total RNA was isolated from the cells, and
quantitative RT-PCR was performed, using oligo(dT) as the RT
primer. IFN.beta. mRNA was quantified and normalized to
.beta.-actin mRNA levels.
DETAILED DESCRIPTION
[0035] A handful of VLP-based vaccines are currently commercialized
worldwide: GlaxoSmithKline's Engerix (hepatitis B virus) and
Cervarix (human papillomavirus), and Merck and Co., Inc.'s
Recombivax HB (hepatitis B virus) and Gardasil (human
papillomavirus) are some examples. Formed by expression of viral
structural proteins, but lacking genetic material that would allow
replication, VLPs are safe, in that they cannot cause infection.
VLPs are also immunogenic, presenting antigens nearly identical to
those presented by authentic virus infection, to elicit both B cell
and T cell responses. A limitation of VLPs, including commercially
available VLP-based vaccines, is that they often require multiple
immunizations and/or administration in combination with adjuvants.
Therefore, strategies that augment VLP immunogenicity and/or make
co-administration with an adjuvant optional, such as those provided
herein, represent a significant advance in vaccine technology.
[0036] Ebola virus (EBOV) is a member of the filovirus family of
enveloped, negative-sense RNA viruses. The filovirus family is
currently divided into 3 genera of which two, Ebolavirus and
Marburgvirus, include zoonotic pathogens that cause periodic
outbreaks with high fatality rates in humans. Members of the
Ebolavirus genus that have caused multiple outbreaks of severe
disease include EBOV, Sudan virus (SUDV) and Bundibugyo virus
(BDBV). The sole species of the Marburgvirus genus, Marburg virus
(MARV), has caused similar lethal outbreaks. The West Africa EBOV
epidemic in 2013-2016 resulted in >28,000 human infections and
>11,000 deaths, emphasizing the need for effective interventions
to prevent and treat filovirus disease. No filovirus vaccine has
been licensed for human use. Therefore, there remains a need to
continue development of alternate vaccine platforms. Given that the
preferred strategy for Ebola virus vaccination is to vaccinate
people in the context of ongoing outbreaks, for example, by
targeting contacts of patients and where there is imminent risk of
exposure to the virus, vaccines that induce rapid immunity would be
optimal.
[0037] The EBOV matrix protein, VP40, is able to bud from cells to
form filamentous membrane-bound particles that resemble authentic
EBOV. Co-expression with the viral Ebola virus glycoprotein (GP)
results in increased EBOV VLP (eVLP) production with GP present on
the surface of the eVLP membrane. Co-expression of the Ebola viral
nucleoprotein (NP) with VP40 also enhances budding and results in
incorporation of NP into the eVLPs. The combination of VP40, NP and
GP yields eVLPs with GP on the surface and VP40 and NP within the
eVLP membrane. As described herein, eVLPs that elicit a robust
innate immune response, through either the RIG-I or
TIR-domain-containing adapter-inducing interferon-.beta.
(TRIF)-dependent pathways were made. These eVLPs cause
substantially enhanced humoral and cellular immunity. This
exemplary approach involved appending the two CARD domains of
RIG-I, which constitute a functioning signaling domain, onto EBOV
NP C-terminal residues 601-739 (NP.sub.CT), a domain that is
sufficient for incorporation into eVLPs. When the RIG-I 2CARD
domain is fused to NP.sub.CT it induces an IFN response, is
incorporated into eVLPs, stimulates IFN responses in dendritic
cells (DCs) and elicits, after a single immunization in the absence
of adjuvant, a robust anti-GP antibody response far greater than
the response seen with standard eVLPs. Thus, the VLP platform
provided herein is used to enhance the speed and robustness of
anti-VLP immune responses against pathogens or cancer antigens.
VLPs
[0038] Provided herein is a VLP comprising a surface protein or
fragment thereof a matrix protein or fragment thereof optionally,
an intra-VLP protein or a fragment thereof; and a polypeptide that
enhances an immune response, wherein the polypeptide is a
polypeptide adjuvant, and wherein the polypeptide is linked to the
surface protein, the matrix protein or the intra-VLP protein.
[0039] As used herein, a virus-like particle or VLP refers to a
particle that includes structural proteins, for example, viral
structural proteins, but is non-infectious and unable to replicate,
as VLPs contain no viral genetic material. VLPs generally include
one or more structural proteins, for example, viral structural
proteins, including but not limited to capsid, coat, matrix,
nucleoprotein, shell, surface and/or envelope proteins, or
particle-forming polypeptides derived from these proteins.
Optionally, the VLP comprises a surface protein or a
particle-forming fragment thereof, a matrix protein or a
particle-forming fragment thereof, and a nucleoprotein or a
fragment thereof. VLPs can form spontaneously upon recombinant
expression of the structural proteins in an appropriate expression
system. The VLP as described herein optionally includes a surface
protein that is a chimeric protein. For example, a bacterial
protein can be fused with the viral surface protein or fragment
thereof.
[0040] As used throughout, the term optional or optionally means
that the subsequently described event or circumstance can or cannot
occur and that the description includes instances where the event
or circumstance occurs and instances where it does not.
[0041] The terms polypeptide, peptide, and protein are used
interchangeably herein to refer to a polymer of amino acid
residues. As used herein, the terms encompass amino acid chains of
any length, including full-length proteins, wherein the amino acid
residues are linked by covalent peptide bonds. In the compositions
and methods provided herein, a particle-forming polypeptide derived
from a particular protein, for example, from a viral or bacterial
protein, can be a full-length polypeptide or a fragment thereof.
Optionally, the polypeptide derived from a particular protein can
be a polypeptide with internal deletions, insertions or
substitutions, which has the ability to form VLPs under conditions
that favor VLP formation. Accordingly, the polypeptide may comprise
the full-length sequence, fragments, truncated and partial
sequences, as well as analogs and precursor forms of the reference
molecule. The term therefore contemplates deletions, additions and
substitutions to the sequence, as long as the polypeptide retains
the ability to form a VLP. Thus, the term includes natural
variations of the specified polypeptide since variations in
proteins often occur between viral or bacterial isolates. The term
also includes deletions, additions and substitutions that do not
naturally occur in the reference protein, as long as the
polypeptide retains the ability to form a VLP. Optionally,
substitutions are those which are conservative in nature, i.e.,
those substitutions that take place within a family of amino acids
that are related in their side chains. Such modifications include
conservative amino acids substitutions, such that the polypeptide
optionally contains one or more conservative amino acid
substitutions. The following groups each contain amino acids that
are conservative substitutions for one another. These groups are
exemplary as other conservative substitutions are known to those of
skill in the art.
[0042] 1) Alanine (A), Glycine (G);
[0043] 2) Aspartic acid (D), Glutamic acid (E);
[0044] 3) Asparagine (N), Glutamine (Q);
[0045] 4) Arginine (R), Lysine (K);
[0046] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine
(V);
[0047] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
[0048] 7) Serine (S), Threonine (T); and
[0049] 8) Cysteine (C), Methionine (M)
[0050] Preferably, the sequences employed to produce VLPs exhibit
at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% sequence
identity to a naturally occurring surface protein, matrix protein
or intra-VLP protein. In each case, where specific nucleic acid or
polypeptide sequences are recited, embodiments comprising a
sequence having at least 80% (e.g. 80%, 85%. 90%, 95%, 99%)
identity to the recited sequence are also provided. Identity or
similarity with respect to a sequence is defined as the percentage
of amino acid residues in the candidate sequence that are identical
(i.e., same residue) with the starting amino acid residues, after
aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity. Methods of alignment
of sequences for comparison are well known in the art. Optimal
alignment of sequences for comparison can be conducted, for
example, by the local homology algorithm of Smith and Waterman
(Adv. Appl. Math. 2:482, 1970), by the homology alignment algorithm
of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the search
for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci.
USA 85:2444, 1988), by computerized implementations of these
algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Ausubel et al., Current Protocols in Molecular Biology
(1995 supplement)).
[0051] Nucleic acids encoding any of the polypeptide sequences
described herein are also provided. The term nucleic acid or
nucleotide refers to deoxyribonucleic acids (DNA) or ribonucleic
acids (RNA) and polymers thereof in either single- or
double-stranded form. Unless specifically limited, the term
encompasses nucleic acids containing known analogues of natural
nucleotides that have similar binding properties as the reference
nucleic acid and are metabolized in a manner similar to naturally
occurring nucleotides. Unless otherwise indicated, a particular
nucleic acid sequence also implicitly encompasses conservatively
modified variants thereof (e.g., degenerate codon substitutions),
alleles, orthologs, SNPs, and complementary sequences as well as
the sequence explicitly indicated. Specifically, degenerate codon
substitutions may be achieved by generating sequences in which the
third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et
al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol.
Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes
8:91-98 (1994)). A nucleic acid sequence encoding a selected
polypeptide can include, but is not limited to, cDNA from viral,
prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or
prokaryotic DNA, and synthetic DNA sequences. The nucleic acid
sequences described herein can be operably linked to each other in
any combination. For example, one or more nucleic acid sequences
can be expressed from the same promoter and/or from different
promoters. As described below, nucleic acid sequences encoding any
of the polypeptides described herein can be included on one or more
vectors.
[0052] As used throughout, a surface protein can be a transmembrane
protein or the transmembrane domain of a transmembrane protein. In
some embodiments, the surface protein comprises a bacterial
protein. The bacterial protein can be a protein or a fragment
thereof normally expressed on the surface of a bacterium, a protein
or fragment thereof that is normally secreted from a bacterium, a
protein or fragment thereof that is normally within a bacterial
membrane, or a protein or a fragment thereof that is normally
intra-bacterial, for example, normally on the surface of
mycobacteria, secreted by a mycobacterium, with a mycobacterial
membrane, or within the cytoplasm of a mycobacterium. In some
embodiments, the surface protein is a viral surface protein or a
chimeric version thereof. For example, the viral surface protein
can be a protein or fragment thereof normally expressed on the
surface of a virus. In some embodiments, the surface protein is a
polypeptide expressed on a cancer cell. For example, the surface
protein can be a protein or a fragment thereof normally expressed
on the surface of a cancer cell. Alternatively, the surface protein
optionally is a viral surface protein fused with a polypeptide
expressed by a cancer cell.
[0053] Optionally, the surface protein comprises a peptide, for
example, an antigen or portion thereof, that induces a B-cell
mediated immune response, i.e., an immune response mediated by
antibody molecules. The antigen can be a viral antigen, a bacterial
antigen, a fungal antigen, a parasitic antigen, or a cancer
antigen. The antigen can contain one or more epitopes (either
linear, conformational or both) that will stimulate a host's
immune-system to make a humoral and/or cellular antigen-specific
response. Generally, a B-cell epitope will include at least about 5
amino acids but can be as small as 3-4 amino acids, and can vary in
length from about 5 to about 20 amino acids. For example, the
epitope can include about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20 amino acids.
[0054] In some embodiments, the surface protein comprises one or
more viral B cell epitopes. In some embodiments, the surface
protein comprises one or more B cell epitopes from one or more
types or strains of a virus. The surface protein optionally
comprises one or more bacterial, fungal, or parasitic B cell
epitopes. In some embodiments, the surface protein comprises one or
more B cell epitopes from one or more types or strains of bacteria,
fungus, or parasite. When the surface protein comprises a viral,
bacterial, fungal, or parasitic protein, the protein sequences can
comprise chimeric polypeptides, for example, an Ebola virus
chimeric protein in which all or part (s) of the Ebola virus
protein sequence is replaced with sequences from other viruses
and/or sequences from other filoviruses strains or other
non-filoviruses or replaced with sequences from bacteria, funguses,
or parasites. Furthermore, the surface protein is optionally
glycosylated.
[0055] In some embodiments, the surface protein comprises a peptide
that targets the VLP to a particular cell or tissue. The targeting
protein or peptide is optionally linked to the surface protein or a
fragment thereof. Targeting of VLPs to a particular cell(s) or
tissue(s) can also be achieved by linking the targeting peptide to
the VLP after VLP assembly or formation. Methods for linking a
targeting peptide to a VLP include, but are not limited to,
chemical crosslinking (Jegerlehner et al. Vaccine 20: 3104-12
(2002)), click chemistry conjugation (Patel et al. Bioconjugate
Chem. 22: 376-87 (2011)), or SpyTag/SpyCatcher reaction (Brune et
al. Sci Rep. 6: 19234 (2016))
[0056] As used throughout, a matrix protein is a polypeptide or a
fragment thereof that promotes assembly of a VLP, optionally, via
interaction(s) with the surface protein and/or intra-VLP protein.
In some embodiments, the matrix protein is a viral matrix protein
or a fragment thereof. Optionally the matrix protein is a chimeric
protein. In some embodiments, the matrix protein is fused to a
heterologous antigen such as a viral protein or a fragment thereof,
a bacterial protein or a fragment thereof, a fungal protein or
fragment thereof, or a parasitic protein or fragment thereof. For
example, the matrix protein is optionally fused to a T-cell
antigen.
[0057] As used throughout, an intra-VLP protein or a fragment
thereof is a polypeptide that is packaged into any of the VLPs
described herein during VLP formation or assembly. An intra-VLP
protein can be incorporated into the lumen of the VLP by budding,
for example, through direct molecular targeting to the VLP or by
means of mass action, wherein the intra-VLP protein is present in
the cytosol of the cell and is non-specifically included in the
fluid that forms the lumen of the VLP upon VLP budding from the
plasma membrane. Although a matrix protein is located inside an
VLP, the term intra-VLP protein or fragment thereof, as used
herein, is not intended to refer to a matrix protein.
[0058] In some embodiments, the intra-VLP protein or fragment
thereof interacts with the matrix protein or surface protein to
promote assembly of a VLP. Optionally, the intra-VLP protein or a
fragment thereof comprises one or more T cell epitopes and induces
a T cell-mediated immune response. Generally, a T cell epitope can
include at least about 7-9 amino acids. Normally, an epitope will
include between about 5 and 20 amino acids, for example, about 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino
acids.
[0059] As used throughout, a T cell-mediated immune response can
include the production of cytokines, chemokines and/or other such
molecules produced by activated T cells and/or other white blood
cells, including those derived from CD4+ and CD8+ T cells. In some
embodiments, the T cell response comprises an antigen-specific
response by helper T cells. Helper T cells act to help stimulate
the function and to focus the activity of nonspecific effector
cells against cells displaying peptide antigens in association with
MHC molecules on their surface. T cells can be activated after
encountering antigen-presenting cells (APCs) (for example,
macrophages, dendritic cells or B cells) that have taken up one or
more of the VLPs described herein and present a T cell antigen on
their surface via association with the major histocompatibility
complex on the APCs. In some embodiments, activating
antigen-specific cytotoxic T cells induce apoptosis in cells
displaying epitopes of a foreign antigen on their surface. These
include, but are not limited to, virus-infected cells, cells with
intracellular bacteria, and cancer cells displaying tumor antigens.
In some embodiments, the intra-VLP protein is a viral nucleoprotein
(NP) or a fragment thereof. The NP or fragment thereof is
optionally linked to a T cell antigen. The intra-VLP protein can be
a C-terminal fragment of a viral NP, for example, a C-terminal
fragment of an Ebola virus NP. In some embodiments the C-terminal
fragment corresponds to amino acid residues 601-739 of the Zaire
Ebola virus strain Mayinga. By way of example the C-terminal
fragment corresponds to
(TPTVAPPAPVYRDHSEKKELPQDEQQDQDHTQEARNQDSDNTQSEHSFEEMYRHIL
RSQGPFDAVLYYHMMKDEPVVFSTSDGKEYTYPDSLEEEYPPWLTEKEAMNEENRF
VTLDGQQFYWPVMNHKNKFMAILQHHQ (SEQ ID NO:1) or a sequence having at
least 85%, 90%, 95%, or 99% identity to SEQ ID NO:1.
[0060] The VLPs provided herein comprise a polypeptide that
enhances an immune response, wherein the polypeptide is a
polypeptide adjuvant. The polypeptide adjuvant optionally enhances
the immune response as compared to the immune response of a VLP
that does not comprise a polypeptide adjuvant, i.e., a
non-adjuvanted VLP. In some embodiments, the polypeptide adjuvant
enhances the immune response by reducing the number of
immunizations necessary to stimulate a lasting immune response, for
example, a protective response against a pathogen. In some
embodiments, the polypeptide adjuvant is linked to the surface
protein, the matrix protein or the intra-VLP protein. Optionally,
the polypeptide adjuvant is linked to the surface protein, the
matrix protein or the intra-VLP protein via a peptide linker, for
example, by a Ser-Gly linker (e.g., SGGSGGSG (SEQ ID NO:2).
Additional linkers are described in Klein et al. (2014) Design and
characterization of structured protein linkers with differing
flexibilities, Protein Eng. Des. Sel. 27(10):325-330, which is
incorporated by reference herein in its entirety. Optionally, the
peptide linker is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length.
[0061] By incorporating a polypeptide adjuvant that enhances an
immune response, the VLPs provided herein are self-adjuvanted VLPs.
By incorporating an adjuvant into the VLPs, the amount of an
adjuvant that is not incorporated into the VLP, i.e., a non-VLP
adjuvant administered to the subject can be reduced or obviated
when the VLPs are administered to a subject. For example, the
amount of a non-VLP adjuvant can be reduced by at least 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In some embodiments, the
self-adjuvanted VLPs described herein reduce the amount of antigen
required for stimulating an immune response. It is understood that,
although the VLPs provided herein can be administered without a
non-VLP adjuvant, the methods provided herein include
co-administration of any of the VLPs provided herein with a non-VLP
adjuvant. Similarly, the VLP compositions provided herein can
comprise the polypeptide adjuvant and optionally further comprise
an adjuvant that is packaged into the VLP but is not linked to the
matrix protein, the surface protein or the intra-VLP protein
non-VLP adjuvant.
[0062] The self-adjuvanted VLPs provided herein can be used to
enhance an anti-pathogenic immune response (for example, an
antiviral, antibacterial, anti-fungal, or anti-parasitic immune
response) or to enhance an anti-cancer immune response. In some
embodiments, the polypeptide adjuvant enhances a B-cell mediated
immune response, a T cell-mediated immune response, or both a
B-cell mediated immune response and a T cell-mediated immune
response. The polypeptide adjuvant optionally induces a Type I
interferon immune response.
[0063] In some embodiments, the polypeptide adjuvant comprises one
or more signaling domains, such as caspase activation and
recruitment domains (CARDs). Optionally, the polypeptide adjuvant
comprises at least one CARD domain from a RIG-I-like receptor, such
as retinoic acid-inducible gene-I (RIG-I). For example, the
polypeptide adjuvant optionally comprises one, two, or more CARDs
of RIG-I or MDA5. In some embodiments, the polypeptide adjuvant
comprises one or more repeats of the amino acid sequence for RIG-I
(MTTEQRRSLQAFQDYIRKTLDPTYILSYMAPWFREEEVQYIQAEKNNKGPMEAATL
FLKFLLELQEEGWFRGFLDALDHAGYSGLYEAIESWDFKKIEKLEEYRLLLKRLQPEF
KTRIIPTDIISDLSECLINQECEEILQICSTKGMMAGAEKLVECLLRSDKENWPKTLKL
ALEKERNKFSELWIVEKGIKDVETEDLEDKMETSDIQ (SEQ ID NO:3)) or one or more
repeats of an amino acid sequence having at least 85%, 90%, 95%, or
99% identity with SEQ ID NO:3. In some embodiments, the polypeptide
adjuvant comprises one or more repeats of the amino acid sequence
for MDA5 (MSNGYSTDENFRYLISCFRARVKMYIQVEPVLDYLTFLPAEVKEQIQRTVATSGNMQ
AVELLLSTLEKGVWHLGWTREFVEALRRTGSPLAARYMNPELTDLPSPSFENAHDEY
LQLLNLLQPTLVDKLLVRDVLDKCMEEELLTIEDRNRIAAAENNGNESGVRELLKRIV
QKENWFSAFLNVLRQTGNNELVQELTGSD (SEQ ID NO:4)) or one or more repeats
of an amino acid sequence having at least 85%, 90%, 95%, or 99%
identity with SEQ ID NO:4.
[0064] In some embodiments, the viral surface protein or a fragment
thereof, the viral matrix protein or a fragment thereof and the
viral NP or a fragment thereof are from the same virus. The virus
can be a virus that infects human or non-human animals. It is
understood that the virus can also be the animal counterpart to any
human virus described herein. All strains and types and subtypes of
each virus described herein are also contemplated. In some
embodiments, the viral surface protein or a fragment thereof, the
viral matrix protein or a fragment thereof, and the viral
nucleoprotein or a fragment thereof are from one or more of a of a
filovirus, an arenavirus, a paramyxovirus (e.g., a henipavirus),
pneumovirus (e.g., respiratory syncytial virus), and an influenza
virus. Additionally, the viral surface protein or a fragment
thereof, the viral matrix protein or a fragment thereof, and the
viral nucleoprotein or a fragment thereof or their corresponding
proteins from one or more of Norovirus; Hepatitis C virus;
alphavirus (e.g. eastern equine encephalitis virus, Venezuelan
equine encephalitis virus, Western equine encephalitis virus, and
Mayaro virus); Bunyavirus (e.g. Rift Valley fever virus, Crimean
Congo hemorrhagic fever virus, Severe fever with thrombocytopenia
syndrome virus (SFTSV)); and rhabdoviruses (vesicular stomatitis
virus, rabies virus, lyssaviruses) could similarly be used.
Flaviviruses include, but are not limited to, West Nile virus,
Dengue virus (types 1 to 4), yellow fever virus, Japanese
encephalitis virus and Zika virus. Arenaviruses include, but are
not limited to, lymphocytic choriomeningitis virus, Lujo virus,
Lassa fever virus, Machupo virus, Savia virus and Whitewater Arroyo
virus. Henipaviruses include, but are not limited to, Hendra virus
and Nipah virus. Influenza viruses include, but are not limited to,
Influenza virus A, B, C and D. All strains and subtypes of
influenza virus are also contemplated, including but not limited
to, H1N1, H2N2, H3N2, H7N9, and H5N1 influenza A viruses.
[0065] In some embodiments, the viral surface protein is an Ebola
virus glycoprotein (GP) or a fragment thereof, the viral matrix
protein is Ebola virus VP40 matrix protein or a fragment thereof,
and the viral NP is an Ebola NP or a fragment thereof. In some
embodiments, the Ebola virus GP, the Ebola virus VP40 matrix
protein, or the Ebola virus NP or a fragment thereof is linked to a
polypeptide adjuvant comprising one or more signaling domains
(e.g., CARDs) as described herein.
[0066] The viral surface protein is optionally an influenza virus
GP or a fragment thereof, the viral matrix protein is optionally an
influenza virus M1 protein or a fragment thereof and the viral NP
is optionally an influenza virus NP or a fragment thereof. In some
embodiments, the influenza virus GP or a fragment thereof, the
influenza virus M1 protein or a fragment thereof or the influenza
NP or a fragment thereof is linked to a polypeptide adjuvant
comprising one or more signaling domains (e.g., CARDs).
[0067] In some embodiments, the viral surface protein is Lassa
virus glycoprotein complex (GPC) or a fragment thereof, the viral
matrix protein is Lassa virus matrix protein Z or a fragment
thereof, and the viral NP is a Lassa virus NP or a fragment
thereof. Optionally, the Lassa virus GPC or a fragment thereof, the
Lassa virus matrix protein Z or a fragment thereof, or the Lassa
virus NP or a fragment thereof is linked to a polypeptide adjuvant
comprising one or more signaling domains (e.g., CARDs).
[0068] In some embodiments, the viral surface protein is a Nipah
virus glycoprotein G or a fragment thereof or a Ninah virus fusion
(F) protein or a fragment thereof, the viral matrix protein is a
Nipah virus matrix (M) protein or a fragment thereof and the viral
NP is a Nipah NP or a fragment thereof. Optionally, the Nipah virus
glycoprotein F protein or a fragment thereof, the Nipah virus M
protein or a fragment thereof or the Nipah virus NP or a fragment
thereof is linked to a polypeptide adjuvant comprising one or more
signaling domains (e.g., CARDs).
[0069] In some embodiments, the surface protein or a fragment
thereof, the viral matrix protein or a fragment thereof, and the
viral nucleoprotein or a fragment thereof are from different
families of the same virus or from different viruses. For example,
and not to be limiting, in some embodiments, the VLP can comprise a
viral matrix protein or a fragment thereof from a first virus (for
example, an Ebola virus VP40 matrix protein), a surface protein or
a fragment thereof from a second virus, and/or in intra-VLP protein
or a fragment thereof from a third virus. Optionally, the first,
second and third viruses are selected from the group consisting of
a Filovirus, an arenavirus, a paramyxovirus (e.g., a henipavirus),
a pneumovirus (e.g., respiratory syncytial virus), and an influenza
virus. Table 1 shows various combinations.
TABLE-US-00001 TABLE 1 Matrix protein nucleoprotein glycoprotein
VP40 Ebola virus NP Ebola virus GP VP40 other filovirus NP other
filovirus GP VP40 Ebola virus NP Arenavirus GP VP40 Ebola virus NP
paramyxovirus (including henipaviruses) glycoproteins VP40 Ebola
virus NP pneumovirus (including respiratory syncytial virus)
glycoproteins VP40 Ebola virus NP influenza virus hemagglutinin and
or neuraminidase VP40 Ebola virus NP chimeric Ebola virus/other
virus glycoprotein to enhance incorporation into VLPs Nipah virus
Nipah virus Nipah virus fusion (F) matrix protein nucleoprotein
and/or glycoprotein (G) (M) Respiratory Respiratory syncytial
Respiratory syncytial virus syncytial virus virus nucleoprotein
fusion and/or glycoprotein matrix protein Other Other paramyxovirus
Other paramyxovirus fusion paramyxovirus nucleoprotein and/or other
paramyxovirus matrix (M) glycoprotein Lassa virus Z Lassa virus
Lassa virus glycoprotein protein nucleoprotein Other arenavirus
Other arenavirus Other arenavirus Z protein nucleoprotein
glycoprotein
[0070] The VLP can comprise a surface protein (e.g., a viral
protein, a bacterial protein, a fungal protein, a parasitic
protein, or a fragment thereof), a viral matrix protein (for
example, an Ebola virus VP40 matrix protein), and a viral intra-VLP
protein (for example, an Ebola virus NP) or a fragment thereof. In
some embodiments, the surface protein or a fragment thereof, the
viral matrix protein or a fragment thereof, or the viral intra-VLP
protein or a fragment thereof is linked to a polypeptide adjuvant
comprising one or more signaling domains (e.g., CARDs).
[0071] Optionally, the surface protein is a cancer antigen, the
matrix protein is a viral matrix protein (for example, Ebola virus
VP 40 matrix protein) or a fragment thereof and the intra-VLP
protein is a viral nucleoprotein (for example, Ebola virus NP) or a
fragment thereof. In some embodiments, the cancer antigen, the
viral matrix protein or a fragment thereof, or the viral intra-VLP
protein or a fragment thereof is linked to a polypeptide adjuvant
comprising one or more CARDs.
[0072] Optionally, the VLP directed to a bacterial target comprises
a bacterial protein or a fragment thereof, a matrix protein or a
fragment thereof, and an intra-VLP protein or a fragment thereof.
The VLP can comprise a viral surface protein; 2CARDs, optionally
fused to a matrix protein or an intra-VLP protein; and a bacterial
protein, optionally fused to a matrix or intra-VLP protein.
Expression Vectors and Host Cells
[0073] Sequences encoding the desired polypeptides to be
incorporated into any of the VLPs described herein can be cloned
into any suitable vector or replicon for expression. Numerous
cloning vectors are known to those of skill in the art, and one of
skill in the art can readily select appropriate vectors and control
elements for any given host cell type in view of the present
specification as well as information known in the art about
expression (see Ausubel et al. Current Protocols in Molecular
Biology, Wiley & Sons; and Green, 1988; and Sambrook Molecular
Cloning--A Laboratory Manual, 4th Ed., Cold Spring Harbor
Laboratory Press, New York (2001)).
[0074] Non-limiting examples of vectors that can be used to express
sequences that assemble into VLPs include viral-based vectors (for
example, retrovirus, adenovirus, adeno-associated virus,
lentivirus), baculovirus vectors, plasmid vectors, non-viral
vectors, mammalian vectors, mammalian artificial chromosomes, and
combinations thereof.
[0075] Host cells can be transformed, transfected, or infected with
one or more expression vectors containing nucleic acid encoding the
polypeptides, under conditions and for an amount of time sufficient
to allow expression of the proteins and formation of VLPs. Such
conditions for protein expression vary with the choice of the
expression vector and the host cell and are ascertainable by one
skilled in the art through routine experimentation.
[0076] Expression vectors typically contain coding sequences and
expression control elements that allow expression of the coding
regions in a suitable host. For example, sequence(s) encoding the
polypeptide(s) can be inserted into an expression vector that
contains transcriptional and translational regulatory sequences,
which include, e.g., promoter sequences, ribosomal binding sites,
transcriptional start and stop sequences, translational start and
stop sequences, transcription terminator signals, polyadenylation
signals, and enhancer or activator sequences. In addition, the
expression vector can include more than one replication system,
such that it can be maintained in two different organisms, for
example, in mammalian or insect cells for expression and in a
prokaryotic host for cloning and amplification.
[0077] Typical promoters used for mammalian cell expression include
the SV40 early promoter, a CMV promoter (such as the CMV immediate
early promoter, optionally including intron A), RSV, HIV-LTR, the
mouse mammary tumor virus LTR promoter (MMLV-LTR), FIV-LTR, the
adenovirus major late promoter (Ad MLP), and the herpes simplex
virus promoter, among others. Other nonviral promoters, such as a
murine metallothionein promoter can also be used for mammalian
expression. Enhancer elements can also be used to increase
expression levels of the constructs, for example, in mammalian host
cells. Examples include the SV40 early gene enhancer, the
enhancer/promoter derived from the long terminal repeat (LTR) of
the Rous Sarcoma Virus, and elements derived from human CMV, to
name a few.
[0078] It is understood that an expression vector may contain one
or more nucleic acid sequences as described herein. For example, in
some embodiments, a single vector comprises sequences encoding all
of the proteins found in a VLP. Alternatively, multiple vectors may
be used (for example, multiple constructs, each encoding a single
polypeptide-encoding sequence or multiple constructs, each encoding
one or more polypeptide-encoding sequences). In embodiments in
which a single vector comprises multiple polypeptide-encoding
sequences, the sequences may be operably linked to the same or
different transcriptional control elements within the same
vector.
[0079] The ability of a surface protein, a matrix protein, and an
intra-VLP protein or fragments thereof to self-assemble into VLPs
with antigenic proteins presented on the surface allows these VLPs
to be produced in any host cell by the co-introduction of the
nucleic acid sequences encoding the surface protein, matrix
protein, and the intra-VLP protein or fragments thereof. In some
embodiments, the sequence(s) are stably integrated into a host
cell. In some embodiments, the sequence(s) are transiently
integrated into a host cell. Suitable host cells include, but are
not limited to, bacterial, yeast, insect, Xenopus, avian,
mammalian, and plant cells. Host cells that comprise or secrete any
of the VLPs described herein are also provided. Host cells that
express sequences described herein to produce self-assembly of VLPs
are also provided herein. Populations of any of the host cells
described herein are also provided.
Methods of Making VLPs
[0080] Provided herein is a method of making any of the VLPs
described herein. The method comprises the steps of (a) expressing
in a host cell at least one VLP described herein; (b) growing the
host cell under conditions which allow the formation of VLPs; and
(c) purifying the VLPs. Also provided is a method of making an
immunogenic composition comprising any of the VLPs described herein
and a pharmaceutically acceptable carrier. The method comprises (a)
expressing in a host cell at least one VLP described herein; (b)
growing the host cell under conditions which allow the formation of
VLPs; (c) purifying the VLPs; and (d) preparing the immunogenic
composition with the purified VLPs (e.g., by adding a
pharmaceutically acceptable carrier). An immunogenic composition
comprising at least one VLP produced by any of the methods
described herein is also provided.
[0081] In some embodiments, the host cell is transfected or
infected with one or more recombinant constructs encoding (a) the
surface protein; (b) the matrix protein, (c) the intra-VLP protein
or fragments thereof; and (d) a polypeptide that enhances an immune
response, wherein the polypeptide is linked to the surface protein,
the matrix protein or the intra-VLP protein or fragments thereof
when expressed in the host cell.
[0082] When expression vectors containing the nucleic acid
sequences encoding the proteins necessary for VLP formation are
introduced into host cell(s) and subsequently expressed, the VLPs
assemble and are then released from the cell surface into the
culture media. Depending on the expression system and host
selected, the VLPs are produced by growing host cells transformed
by an expression vector(s) under conditions whereby the
particle-forming polypeptides are expressed and VLPs can be formed.
The selection of the appropriate growth conditions is within the
skill of the art. If the VLPs accumulate intracellularly, the cells
can be disrupted, using chemical, physical or mechanical means,
which lyse the cells yet keep the VLPs substantially intact. Such
methods are known to those of skill in the art. Alternatively, VLPs
may be secreted and harvested from the surrounding culture media.
See, for example, Kim and Kim, Letters in Applied Microbiology 64:
111-123 (2016)). The particles are then isolated (or substantially
purified) using methods that preserve the integrity thereof, such
as, by density gradient centrifugation, e.g., sucrose gradients,
PEG-precipitation, pelleting, and the like (see, for example,
Vicente et al. J. Invertebr. Pathol. 107: S42-8 (2011); and Peyret
Journal of Virological Methods 225: 59-63 (2015)), as well as
standard purification techniques including, but not limited to, ion
exchange and gel filtration chromatography.
Immunogenic Compositions
[0083] Any of the VLPs described herein or mixtures thereof can be
provided in an immunogenic composition, for example, a vaccine.
These include for example, an immunogenic composition comprising an
effective amount of any of the VLPs described herein or mixtures
thereof and a pharmaceutical carrier. The term carrier means a
compound, composition, substance, or structure that, when in
combination with the VLP(s), aids or facilitates preparation,
storage, administration, delivery, effectiveness, selectivity, or
any other feature of the VLP(s) for its intended use or purpose.
For example, a carrier can be selected to minimize any degradation
of the active ingredient and to minimize any adverse side effects
in the subject. Such pharmaceutically acceptable carriers include
sterile biocompatible pharmaceutical carriers, including, but not
limited to, saline, buffered saline, dextrose, proteins,
polysaccharides, polylactic acids, polyglycolic acids, polymeric
amino acids, amino acid copolymers, lipid aggregates (for example,
oil droplets or liposomes) and water. Typically, the carrier is a
molecule that does not itself induce the production of antibodies
harmful to the subject receiving the composition.
[0084] In some embodiments, the immunogenic composition comprises
an adjuvant that is not incorporated into the VLP. These include,
but are not limited to aluminum salts (for example, aluminum
hydroxide, aluminum phosphate, aluminum sulfate, etc.); muramyl
peptides; bacterial cell wall components; Complete Freunds Adjuvant
(CFA); Incomplete Freunds Adjuvant (IFA); cytokines (for example,
interleukins (IL-1, IL-2, etc.), macrophage colony stimulating
factor (M-CSF), tumor necrosis factor (TNF), beta chemokines (MIP,
1-alpha, 1-beta Rantes, etc.); and detoxified mutants of cholera
toxin (CT), a pertussis toxin (PT), or an E. coli heat-labile toxin
(LT), to name a few.
[0085] Immunogenic compositions comprising any of the VLPs
described herein can be prepared according to standard techniques
and further comprise a pharmaceutically acceptable carrier. These
compositions are usually sterile. The pharmaceutical compositions
can also contain a pharmaceutically acceptable excipient. Such
excipients include any pharmaceutical agent that does not itself
induce an immune response harmful to the individual receiving the
composition, and which may be administered without undue toxicity.
Pharmaceutically acceptable excipients include, but are not limited
to, liquids such as water, saline, glycerol, sugars and ethanol.
Pharmaceutically acceptable salts can be included therein, for
example, mineral acid salts such as hydrochlorides, hydrobromides,
phosphates, sulfates, and the like; and the salts of organic acids
such as acetates, propionates, malonates, benzoates, and the like.
Additionally, auxiliary substances, such as wetting or emulsifying
agents, pH buffering substances, and the like, may be present in
such vehicles. The preparation of pharmaceutically acceptable
carriers, excipients and formulations containing these materials is
described in, e.g., Remington: The Science and Practice of
Pharmacy, 22nd edition, Lloyd V. Allen et al, editors,
Pharmaceutical Press (2012).
[0086] Aqueous solutions can be packaged for use or filtered under
aseptic conditions and lyophilized, the lyophilized preparation
being combined with a sterile aqueous solution prior to
administration. The compositions can contain pharmaceutically
acceptable auxiliary substances as required to approximate
physiological conditions, such as pH adjusting and buffering
agents, tonicity adjusting agents and the like (for example, sodium
acetate, sodium lactate, sodium chloride, potassium chloride, and
calcium chloride). Any lipid containing suspension may include
lipid-protective agents that protect lipids against free-radical
and lipid-peroxidative damages on storage. Lipophilic free-radical
quenchers, such as alpha tocopherol and water-soluble iron-specific
chelators, such as ferrioxamine, are suitable.
[0087] The concentration of the VLPs in the immunogenic
compositions can vary widely, i.e., from about 0.05% to about 30%
by weight and will be selected primarily by fluid volumes and
viscosities, in accordance with the particular mode of
administration selected. Alternatively, the VLPs can be dried or
lyophilized and resuspended to a desired concentration in water or
buffers at the time of use.
Administration
[0088] Provided herein is a method of stimulating an immune
response in a subject comprising administering to the subject an
effective amount of any of the VLPs or mixtures of various VLPs
described herein. Also provided is a method of stimulating an
immune response in a subject comprising administering to the
subject an effective amount of any of the immunogenic compositions
comprising any of the VLPs described herein or a mixture of VLPs
described herein. As used throughout, immunogenic refers to an
agent that stimulates the immune system of a living organism, so
that one or more functions of the immune system are increased and
directed towards the immunogenic agent.
[0089] In some embodiments, a population of VLPs that stimulate an
immune response against a first antigen can be co-administered with
a population of VLPs that stimulate an immune response against a
second antigen or a second epitope or a second epitope on the first
antigen. For example, a population of VLPs that stimulate an immune
response against a first cancer antigen can be co-administered with
a population of VLPs that stimulate an immune response against a
second cancer antigen. For example, a population of VLPs that
stimulate an immune response against a first viral antigen can be
co-administered with a population of VLPs that stimulate an immune
response against a second viral antigen. For example, a first
population of VLPs that contain an antigen, but lack the 2CARD
domain, can be administered with a second population of VLPs that
contain a 2CARD domain, but lack the antigen. It is understood that
the methods are not limited to stimulating immune responses against
a first and second antigen, as several populations of VLPs that
stimulate an immune response against a first, second, third,
fourth, fifth antigen etc., respectively, can be co-administered to
the subject.
[0090] Also provided is a method of preventing or reducing the risk
of pathogenic infection in a subject comprising administering to
the subject an effective amount of any of the VLPs or
VLP-containing immunogenic compositions or mixtures thereof
described herein. When the terms prevent, preventing, and
prevention are used herein in connection with a given treatment for
a given condition (e.g., preventing pathogenic infection), they
mean that the treated subject either does not develop a clinically
observable level of the condition at all, or develops it more
slowly and/or to a lesser degree than the subject would have absent
the treatment. These terms are not limited solely to a situation in
which the subject experiences no aspect of the condition
whatsoever. For example, a treatment will be said to have prevented
the condition if it is given during exposure of a subject to a
stimulus that would have been expected to produce a given
manifestation of the condition, and results in the subject's
experiencing fewer and/or milder symptoms of the condition than
otherwise expected. A treatment can prevent infection by resulting
in the subject's displaying only mild overt symptoms of the
infection; it does not imply that there must have been no
penetration of any cell by the infecting microorganism.
[0091] Similarly, reduce, reducing, and reduction as used herein in
connection with the risk of infection with a given treatment (e.g.,
reducing the risk of pathogenic infection) refers to a subject
developing an infection more slowly or to a lesser degree as
compared to a control or basal level of developing an infection in
the absence of a treatment (e.g., administration of VLPs or a
VLP-containing immunogenic composition). A reduction in the risk of
infection may result in the subject's displaying only mild overt
symptoms of the infection or delayed symptoms of infection
[0092] As used throughout, by subject is meant an individual. The
subject can be an adult subject or a pediatric subject. Pediatric
subjects include subjects ranging in age from birth to eighteen
years of age. Thus, pediatric subjects of less than about 10 years
of age, five years of age, two years of age, one year of age, six
months of age, three months of age, one month of age, one week of
age, or one day of age are also included as subjects. Preferably,
the subject is an animal, for example, a mammal such as a primate,
and, more preferably, a human. Non-human primates are subjects as
well. The term subject includes domesticated animals (such as cats,
dogs, etc.), livestock (for example, cattle, horses, pigs, sheep,
goats, etc.), poultry (e.g., chickens, turkeys, etc.), fish (e.g.,
farm-raised fish), and laboratory animals (for example, ferret,
chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.). Thus,
veterinary uses and medical formulations are contemplated
herein.
[0093] The VLPs or VLP-containing immunogenic compositions are
administered in a number of ways, including, for example, orally,
intranasally, via inhalation, via nebulizer, parenterally,
intravenously, intraperitoneally, intracranially, intraspinally,
intrathecally, intraventricularly, intramuscularly, intratumorally
or near a tumor, subcutaneously, intracavity, or transdermally.
Effective doses for any of the administration methods described
herein can be extrapolated from dose-response curves derived from
in vitro or animal model test systems.
[0094] The VLPs or VLP-containing immunogenic compositions can be
administered prior to, concurrent with, or subsequent to delivery
of other VLPs or immunogenic compositions, for example, a vaccine.
Also, the site of VLP administration may be the same or different
as other vaccine compositions that are being administered. The VLPs
or VLP-containing immunogenic compositions can be administered as a
single dose schedule or as multiple doses. In some embodiments, the
VLPs or the VLP-containing immunogenic compositions induce an
immune response, for example, a protective immune response, with
fewer doses as compared to a non-self-adjuvanted VLP delivered with
an adjuvant or a non-VLP based immunogenic composition delivered
with an adjuvant. In some embodiments, the VLPs or the
VLP-containing immunogenic compositions induce an immune response
with a single dose. In some embodiments, the VLPs or the
VLP-containing immunogenic composition induces a protective immune
response with a single dose. As set forth above, an immune response
can be a humoral immune response and/or cellular immune response
leading to the activation or proliferation of B- and/or
T-lymphocytes and/or antigen presenting cells. In some embodiments,
the immune response is an immune response against a pathogen in the
subject, for example, a virus or a bacterium. Examples of viruses
include a Filovirus, an arenavirus, a paramyxovirus (e.g., a
henipavirus), pneumovirus (e.g., respiratory syncytial virus), and
an influenza virus in the subject. More specifically, the virus can
be an Ebola virus, Nipah virus, Lassa virus or influenza virus in
the subject. In some embodiments, the immune response is an immune
response against a bacterium. Examples of bacteria include
mycobacteria, for example, M. tuberculosis.
[0095] In some embodiments, the immune response is against a cancer
antigen. Examples of cancer or tumor antigens include, but are not
limited to non-mutated shared antigens (e.g., MAGE, BAGE, RAGE, and
NY-ESO), that are expressed in testes and in multiple tumor cells;
differentiation antigens (e.g., prostate-specific membrane antigen
(PSMA) and prostate-specific antigen (PSA) in prostate carcinoma,
Mart1/MelanA and tyrosinase present in many melanoma, and carcino
embryonic antigen (CEA) present in a large percentage of colon
cancers) that are tissue restricted and present in lineage-specific
tumor cell; mutated oncogenes and tumor suppressor genes (e.g.,
mutated ras, rearranged bcr/abl, mutated p 53); oncovirus-derived
epitopes (e.g., the human papillomavirus-encoded E6 and E7
proteins, Epstein-Barr virus (EBV)-associated antigens present in
primary brain lymphoma); and nonmutated oncofetal proteins such as
CEA, .alpha.-fetoprotein, and survivin. In some embodiments, a
cancer antigen expressed by the subject, for example, an antigen
expressed on the surface of a tumor cell in a subject is
identified. After identification of the tumor antigen, VLPs
comprising a surface protein that includes the tumor antigen or an
intra-VLP protein comprising a tumor antigen can be made and
administered to the subject according to the methods described
herein.
[0096] The dosage regimen may be selected in accordance with a
variety of factors including type, species, age, weight, sex,
medical condition of the patient; the severity of the condition to
be treated; the route of administration; the renal and hepatic
function of the patient; and the particular pharmaceutical
composition employed. One of ordinary skill in the art can readily
determine and prescribe the effective amount of the composition
(and potentially other agents including therapeutic agents).
[0097] Ranges may be expressed herein as from about one particular
value, and/or to about another particular value. When such a range
is expressed, another aspect includes from the one particular value
and/or to the other particular value. Similarly, when values are
expressed as approximations, by use of the antecedent about, it
will be understood that the particular value forms another aspect.
It will be further understood that the endpoints of each of the
ranges are significant both in relation to the other endpoint, and
independently of the other endpoint.
[0098] Disclosed are materials, compositions, and ingredients that
can be used for, can be used in conjunction with or can be used in
preparation for the disclosed embodiments. These and other
materials are disclosed herein, and it is understood that when
combinations, subsets, interactions, groups, etc. of these
materials are disclosed that while specific reference of each
various individual and collective combinations and permutations of
these compositions may not be explicitly disclosed, each is
specifically contemplated and described herein. For example, if a
method is disclosed and discussed, and a number of modifications
that can be made to a number of molecules included in the method
are discussed, each and every combination and permutation of the
method, and the modifications that are possible are specifically
contemplated unless specifically indicated to the contrary.
Likewise, any subset or combination of these is also specifically
contemplated and disclosed. This concept applies to all aspects of
this disclosure including, but not limited to, steps in methods
using the disclosed compositions. Thus, if there are a variety of
additional steps that can be performed, it is understood that each
of these additional steps can be performed with any specific method
steps or combination of method steps of the disclosed methods, and
that each such combination or subset of combinations is
specifically contemplated and should be considered disclosed.
[0099] Publications cited herein and the material for which they
are cited are hereby specifically incorporated by reference in
their entireties. The following description provides further
non-limiting examples of the disclosed compositions and
methods.
Examples
VLPs
[0100] To generate Ebola VLPs (eVLPs) capable of triggering a
robust antiviral response, a construct containing the two CARD
domains of RIG-I (2CARD), which is capable of inducing a robust IFN
response upon expression in mammalian cells, was appended to
NP.sub.CT, a region sufficient for interaction with VP40 and
incorporation into eVLPs. A Ser-Gly linker was placed between
NP.sub.CT and RIG-I 2CARD (FIG. 1A). For comparison, constructs
that expressed full-length NP (NP.sub.FL) and NP.sub.CT without the
2CARD fusion were generated. Each construct was hemagglutinin
(HA)-tagged (FIG. 1A). To generate eVLPs, each construct was
transfected into HEK293T cells with plasmids that express
Flag-tagged VP40 and the indicated NP constructs without or with
untagged GP. 48 hours post-transfection, cell supernatants were
harvested and eVLPs pelleted through a 20% sucrose cushion. eVLPs
were resuspended and protein concentrations determined. Endotoxin
levels for all VLPs were less than 0.08 IU (endotoxin units) per
microgram of total protein. By transmission electron microscopy,
all eVLPs displayed the filamentous, curved morphology
characteristic of authentic EBOV, and immunogold staining revealed
GP spikes on the virion surface (FIG. 1B). Western blots of
equivalent amounts of purified eVLPs either left untreated or
treated with trypsin to degrade protein not protected within the
eVLP membrane demonstrated the presence of each of the expressed
viral proteins and incorporation into eVLPs of the NP.sub.CT and
2CARD-NP.sub.CT proteins. As expected, GP was incorporated into
eVLPs when expressed and detection of the surface-exposed GP was
lost upon trypsin treatment (FIG. 1C).
2CARD-NP.sub.CT Induces an IFN Response in HEK293T Cells
[0101] To investigate the capacity of the RIG-I 2CARD domain to
induce a type I IFN response when fused to the C-terminus of Ebola
virus (EBOV) NP, 293T cells were co-transfected with constructs
encoding full-length RIG-I, the 2CARD domain alone, the
2CARD-NP.sub.CT fusion or control empty pCAGGS vector and an
IFN.beta.-firefly luciferase reporter plasmid. A
constitutively-expressed Renilla luciferase reporter plasmid was
used as an internal control. Twenty-four hours post-transfection,
cell lysates were analyzed using a Dual Luciferase Assay (Promega,
Madison, Wis.). As expected, full-length RIG-I did not activate the
IFN.beta. promoter (because no activator, such as RIG-I Activating
virus infection or dsRNA was added), whereas the RIG-I 2CARD
construct, which lacks the regulatory RIG-I helicase domain
activated the IFN.beta. promoter. The 2CARD-NP.sub.CT construct
also exhibited IFN.beta.-inducing capacity (FIG. 2).
2CARD VLPs Activate Enhanced IFN and Cytokine Responses in BMDCs
and Stimulate Robust Antibody Responses after a Single
Immunization.
[0102] To test the hypothesis that introduction of the 2CARD domain
into eVLPs will enhance immune stimulating capacity, mouse bone
marrow-derived DCs (BMDC) were infected with
constructs expressing VP40 alone, VP40+GP, VP40+GP+NP.sub.FL,
VP40+GP+NP.sub.CT, or VP40+GP+2CARD-NP.sub.CT. As a positive
control, the DCs were transfected with polyI:C, an IFN inducer
(FIG. 3A). At 3, 6 and 12 hr post-infection, mRNA was isolated and
quantitative RT-PCR performed. The superior capacity, relative to
the other constructs, of eVLPs containing the 2CARD-NP.sub.CT
fusion to induce expression of IFN-.beta., IFN-stimulated genes
RIG-I and ISG15 and cytokine TNF-.alpha. was demonstrated (FIG.
3A). Because the eVLPs were produced in human cells and tested in
mice, the induced expression is not due to contaminating IFN in the
eVLPs, as human IFN does not signal in mice. Further, C57BL/6 mice
were immunized with a single intraperitoneal (IP) dose of 10 .mu.g
of VP40+GP+NP.sub.FL, VP40+GP+NP.sub.CT, VP40+GP+2CARD-NP.sub.CT or
a high dose (25 .mu.g) of 2CARD eVLP; PBS served as a negative
control. As expected VP40+GP+NP.sub.FL eVLPs, after a single IP
dose, elicited little to no detectable anti-GP antibody (FIG. 3B).
In contrast either 10 .mu.g or 25 .mu.g of the 2CARD eVLPs gave a
robust antibody response (FIG. 2B), demonstrating that the IFN
inducing domain can enhance immunogenicity. These data demonstrate
that enhanced VLPs, for example, EBOV VLPs that incorporate the
IFN-inducing 2CARD domain through fusion to NP.sub.CT, can be
made.
[0103] These data demonstrate that enhanced EBOV VLPs that
incorporate the IFN-inducing 2CARD domain through fusion to
NP.sub.CT can be made.
Mapping Regions for RIG-IN Incorporation into VLP
[0104] For the studies described above, NP.sub.601-739 was
initially fused to RIG-I 2CARD (FIG. 1). Structural studies suggest
that NP residues 645-739 form a distinct, folded C-terminal domain
(CTD). Therefore, a series of 10 amino acid deletions, from 601
towards 645, are constructed to determine whether the region of NP
necessary for interaction with VP40 and incorporation into VLPs can
be shortened, as assessed by coimmunoprecipitation (coIP). Each NP
construct is HA-tagged at its N-terminus and Flag-tagged VP40 is
used in all samples. The results are assessed by quantitative
Western blot to determine minimal regions needed for efficient
interaction with VP40, efficient incorporation into VLPs and for
the capacity to enhance VLP release. Controls include no NP,
full-length NP and the Flag-tagged NP.sub.CT lacking the RIG-IN
fusion. Constructs are assessed in terms of interaction with VP40,
incorporation into VLP, IFN inducing capacity of the fusions upon
expression in 293T cells and IFN induction in the context of VLPs,
as described above.
Determine Whether Larger Fragments of NP can be Fused to RIG-IN and
Still be Efficiently Incorporated into VLPs
[0105] Although NP.sub.601-739 is sufficient to mediate
incorporation of the RIG-IN fusion into VLPs, it is not clear if
additional NP sequences might be either beneficial or detrimental.
From a vaccine development perspective, additional NP sequences
could prove beneficial, given that NP possesses dominant CD4+ and
CD8+ T cell epitopes recognized during human infection with Ebola
virus. Therefore, NP constructs with increasing amounts of
N-terminal sequence (i.e. 601-739, 551-739, 501-739, 451-739) with
or without RIG-IN (but each possessing an N-terminal HA tag) are
compared for interaction with VP40, incorporation into VLP, IFN
inducing capacity of the fusions upon expression in 293T cells and
IFN induction in the context of VLPs, as described above.
Compare Enhanced VLPs Using RIG-IN Versus TRIF as an IFN-Inducing
Domain
[0106] RIG-IN is chosen as an IFN-inducing module because it
represents the N-terminus of a protein that could be fused to the
C-terminus of NP. However, it is not certain that RIG-IN is the
optimal approach. For comparison, the TLR3 adaptor protein TRIF is
fused to NP.sub.CT, with a Ser-Gly linker between the two fusion
partners. VP40 interaction, VLP incorporation and IFN inducing
capacity of the fusions is assessed as above.
Assess Strategies to Produce Enhanced VLPs with Different Filovirus
GPs and NP Sequences
[0107] EBOV, Sudan virus (SUDV), Bundibugyo virus (BDBV) and
Marburg virus (MARV) are the filoviruses for which vaccines are
most needed. Whether the 2CARD-NP.sub.CT eVLP platform efficiently
incorporate SUDV, BDBV and MARV GPs is examined. VLPs are produced
and GP incorporation of the gradient purified VLPs is assessed by
SDS-PAGE and Coomassie Blue staining, as described in Martinez et
al. Virology 264(2): 342-54 (2007)). In addition to using untagged
GP constructs, a version with a HA-tag on the cytoplasmic tail of
these type I transmembrane proteins is made to compare GP
incorporation by anti-Flag Western blot. If incorporation is
comparable to that of EBOV GP, these are compared to the EBOV
2CARD-NP.sub.CT eVLPs in the assays described above.
[0108] To match NP sequences for the virus being targeted, whether
the 2CARD-NP.sub.CT fusions can be swapped while retaining the EBOV
VP40 is determined. EBOV, SUDV and BDBV NP constructs are likely to
work with EBOV VP40 because the CTD of NPs from members of the
Ebolavirus genus are well-conserved. Because the MARV NP CTD is
divergent, a completely MARV-based system (2CARD-NP.sub.CT+VP40+GP
vs NP, VP40, GP MARV VLPs) is also evaluated. All of these
constructs are assessed as described above.
Stimulating Dendritic Cells and Eliciting Immune Responses In
Vivo
[0109] As a measure of the potential for the enhanced VLPs
described herein (for, example, VLPs comprising an IFN-inducing
2CARD domain) to stimulate human innate and adaptive immune
responses, their impact on human DC maturation is examined. By
delivering a robust IFN-inducing domain into the cytoplasm, the
VLPs described herein likely can induce a more robust stimulation
of DCs and more closely mimic virus infection, triggering robust DC
maturation. To verify this, immature human DCs from buffy coats of
anonymous human blood donors are prepared. Primary human immature
DCs are mock treated or treated with standard eVLPs, enhanced eVLPs
or infected with Sendai virus (strain Cantell), a potent activator
of RIG-I signaling, IFN production and DC maturation. Measurements
of DC responses are performed as described in Yen et al. J. Virol.
88(21): 12500-10 (2014); and Yen et al. J. Virol. 90(10): 5108-18
(2016). A Nanostring instrument and the human nCounter Myeloid
Innate Immunity panel, which measures expression of 770 genes in 19
different pathways and processes across 7 different myeloid cell
types are used to measure gene expression. Gene expression
measurements are validated with select measurements of secreted
proteins using cytometric bead assay (BD Pharmingen, Franklin Lake,
N.J.) to measure inflammatory cytokines and chemokines including
IL-6, IL1.beta., IL-12p40, TNF, RANTES and MIP-1.alpha., because
secretion of these was seen in prior eVLP assays. Release of
IFN.beta. and IFN.alpha. are also measured by ELISA (PBL Assay
Science, Piscataway, N.J.). Upregulation of MHC class I and class
II and other cell surface markers of DC maturation (CD40, CD80 and
CD86) is also assessed. Additionally, the capacity of the stimuli
to promote DC-induced stimulation of allogeneic T cell responses is
assessed.
Comparison of Adaptive Immune Responses to Enhanced eVLPs with
Adaptive Immune Responses Elicited by Standard eVLPs
[0110] Three immunizations with standard, unadjuvanted eVLPs were
able to confer protection of BALB/c mice and elicit robust antibody
responses. Antibody and T cell responses of 2CARD-NP.sub.CT eVLPs
and three additional constructs exhibiting a range of IFN-inducing
capacities to standard eVLPs (NP+VP40+GP) after one, two and three
immunizations of BALB/c mice are compared. The goal is to identify
an enhanced eVLP preparation that achieves, in a single
immunization, antibody titers comparable to three doses of standard
eVLPs. Immunizations are 21 days apart. 5 animals are used per
group for longitudinal antibody studies (with bleeds before each
immunization) with 3 animals per group for each T cell assay time
point.
ELISA Assays to Measure Anti-GP Antibody Responses
[0111] ELISA assays for anti-GP antibodies are performed as in FIG.
3B. ELISA plates are coated with recombinant EBOV GP at 1 .mu.g/mL
(rGPd.TM., IBT Bioservices, Rockville, Md.) in phosphate-buffered
saline (PBS). Sera are diluted two-fold starting at 1:100, and
horseradish peroxidase (HRP) conjugated goat anti-mouse IgG
antibody (EMD Millipore, Burlington Mass.) serve as a secondary
antibody. ELISAs are developed using SigmaFast OPD (Sigma, St,
Louis, Mo.) and measured on a PerkinElmer EnVison (Waltham, Mass.)
plate reader at OD of 450 nm. The absorbance cut-offs are defined
as the background+0.1 O.D.
Neutralization Assays
[0112] Vesicular stomatitis viruses (VSV) in which the VSV
glycoprotein (G) is replaced by the EBOV GP serves as a useful
biosafety level 2 surrogate for EBOV neutralization assays, with
the VSV-GP-based assay highly correlating with neutralization
performed with authentic EBOV (Regules et al. N. Engl. J. Med.
376(4): 330-41 (2017); and Konduru et al. J. Virol. Methods 254:1-7
(2018)). A VSV-GP that also encodes, as an extra viral gene, green
fluorescence protein (GFP) is also used. This VSV-GFP-GP virus is
also used for neutralization assays, as described in Duehr et al.
J. Virol. 91(16) (2017). Pre-bleed sera are included as negative
controls. Neutralizing anti-GP mouse mAb 1F8 are used as a positive
control.
T Cell Responses
[0113] A peptide restimulation assay is used to evaluate CD4 and
CD8 T cell responses at 2 weeks post-VLP vaccination, as assessed
by interferon (IFN)-.gamma. production. The peptides are Zaire
Ebola virus BALB/c T cell epitopes for GP (LYDRLASTV
(G.sub.161-169)) (SEQ ID NO: 5) and VP40, for example, (YFTFDLTALK
(VP.sub.171-180) (SEQ ID NO: 6) and PEYMEAIYPVRSNST (VP.sub.11-25))
(SEQ ID NO: 7). For NP, the reported mouse epitopes lie outside the
NP.sub.CT domain used in the studies described above. Therefore,
these epitopes are tested in studies evaluating 2CARD fusions to
VP40 rather than NP. Splenocytes isolated at 2 weeks
post-vaccination are cultured for 5 h in RPMI 1640 with or without
1 .mu.M synthetic peptide and 10 .mu.g/ml brefeldin A is added for
the final 2 h. The cells are blocked with mAbs to FcRIII/II and
stained with anti-CD8 CyChrome, or anti-CD4 CyChrome (BD
Pharmingen) with brefeldin A. Following fixation with 1%
formaldehyde, the cells are permeabilized with 0.5% saponin,
stained with anti-IFN-.gamma. PE (BD Pharmingen), and analyzed by
flow cytometry on a SONY SH800 FACS (Champaign, Ill.). A >2-fold
increase in the frequency of IFN-.gamma.-positive cells, as
compared to irrelevant peptide or no peptide, is considered
positive.
Proliferation and Cytokine Production
[0114] Splenocytes obtained at 2 weeks post-vaccination are assayed
for proliferation by culturing in RPMI 1640 medium in the absence
or presence of 10 .mu.g/ml eVLP. After 5 days in culture, aliquots
of culture supernatants are removed for cytokine analysis and 1
.mu.Ci of [.sup.3H]thymidine is added to each well, with .sup.3H
incorporation determined after 18 h incubation. The levels of
cytokines in the supernatants of the restimulated cells are
determined using a cytometric bead assay (BD Pharmingen) to detect
IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IFN-.gamma., and
TNF-.alpha..
T Cell Enzyme-Linked Immunosorbent Spot-Forming Cell Assay
(ELISPOT) Assays.
[0115] T cell memory responses are assessed by using a "cultured
IFN-.gamma. ELISPOT" approach, which reveals potential
IFN-.gamma.-secreting T cell capacity within a lymphocyte
population (Todryk et al., 128(1): 83-91 (2009). Lymphocytes
isolated from spleen and mediastinal LN are cultured in RPMI with
peptides consisting of CD8 and CD4 epitopes of GP and VP40 as
detailed in the peptide restimulation assay above, as well as full
length recombinant GP protein (IBT Bioservices). Cultures are
supplemented with 100 U/ml IL-2 on days 3 and 7. On day 9, the
cells are washed and rested overnight, before performing an
IFN-.gamma. ELISPOT assay (Mabtech, Cincinnati, Ohio). Medium-only,
irrelevant peptide and phytohaemagglutinin controls are included.
The results are compared with a standard ex vivo IFN-.gamma.
ELISPOT run in parallel, where culture with antigen is only
overnight rather than over 9 days. The ELISPOT assays are conducted
at 4 days, 4 weeks, 4 months post-vaccination, to examine
durability of responses.
B Cell Memory Responses
[0116] Memory B cell responses are studied using a modification of
the limiting dilution assay (Ndungu et al. PLoS Pathog.
5(12):e1000690 (2009); and Slifka et al. J. Immunol. Methods
199(1): 37-46 (1996)). Replicates of three-fold dilutions of cell
suspensions of spleen and bone marrow are cultured for 6 days in
Iscove's medium containing R595 lipopolysaccharide (Sigma),
1.times.10.sup.6 irradiated naive splenocytes, and 1/10th volume
supernatant from concanavalin A-stimulated splenocytes. Cells are
harvested and transferred to 96-well Multi-screen HA Nitrocellulose
filtration plates (Millipore) coated with 50 .mu.l of 2.5 .mu.g/ml
recombinant GP diluted in PBS and an ex-vivo ELISpot assay for
GP-specific antibody-secreting cells performed according to the
MabTech (Cincinnati, Ohio) ELISpot protocol.
Prime-Boost Immunization Strategy
[0117] Mice are vaccinated by the IP route with 1 or 10 .mu.g of
standard or enhanced eVLPs. In a prior study using a prime-boost
approach with standard eVLPs it was found that a 10 .mu.g dose was
protective after 2 boosts but 1 .mu.g was not (protection with
fewer immunizations was not reported). A prime-boost with a lower
dose of enhanced eVLPs is selected for protection. For these
studies, control mice receive PBS as a vaccine. The control PBS
group and the VLP immunized groups consist of 5 or 10 mice,
respectively. Priming occurs on day 0 and boosting on day 21. Mice
are challenged 6 weeks post vaccination by IP injection with 1,000
plaque forming units (a 100% lethal dose) of mouse-adapted EBOV
(maEBOV). Post-challenge, mice are observed twice daily for illness
and serum viremia on day 4, the typical peak day of viremia.
Viremia is assessed both by performing quantitative RT-PCR for
viral genome numbers and by plaque assay on Vero E6 cells. Mice are
weighed daily following challenge and observed for clinical
symptoms according to an approved scoring sheet (ruffled fur,
slowing activity, loss of body conditions, labored breathing,
hunched posture, bleeding, paralysis). Mice are monitored for up to
28 days post-challenge to look for signs of delayed illness. For
all animals that reach humane endpoints, blood is obtained and
liver, spleen, lung, kidney and brain are harvested. For blood,
viral RNA levels are assayed by quantitative RT-PCR, and viral
titers are measured by plaque assay. Other tissues are assessed for
viral antigen by immunohistochemistry, for viral RNA by
quantitative RT-PCR and for viral titers by plaque assay. ELISAs to
measure antibodies to virions and to GP are performed on sera
obtained prior to challenge, obtained on day 4 post-challenge and
in survivors 28 days post-challenge.
Testing Optimized, Enhanced eVLPs for Protection after a Single
Immunization
[0118] A single dose of enhanced eVLPs sufficient to elicit anti-GP
antibody responses that is comparable to three immunizations with
standard eVLPs is determined. These experiments follow the
procedures described above, except that challenge will occur on day
28 post-vaccination.
Incorporation of a Bacterial Antigen into Interferon-Inducing
eVLPs
[0119] Mycobacterium tuberculosis (Mtb) is the causative agent of
tuberculosis. A vaccine effective against pulmonary tuberculosis is
needed. The use of an Ebola VLP vaccine approach described herein
to deliver an Mtb antigen in such a form that the immune enhancing,
interferon-inducing 2CARD domain will also be present in the VLPs,
was studied. For this experiment, the Mtb protein, ESAT6, was
chosen. as this is a known antigen that has been used on other
vaccine platforms. The innovation with this approach is the use of
an enhanced 2CARD VLP as an Mtb vaccine. VLPs were generated by
fusing ESAT6 to EBOV VP40 (ESAT6-VP40) (FIG. 6A) and a
corresponding 2CARD-ESAT6-eVP40. To test whether these VLPs can
stimulate innate immune responses upon infection of cells, the
following experiment was performed.
[0120] VLPs were produced by transfecting the following plasmids
into HEK293T cells: GP+eVP40; GP+eVP40+2CARD-NP.sub.CT;
GP+ESAT6-eVP40; GP+2CARD-ESAT6-eVP40; 2CARD-ESAT6-eVP40. After
harvesting the VLPs, the VLPs were added to HEK293T cells. As a
control, the HEK293T cells were mock treated. Activation of innate
immune responses were then determined by measuring levels of mRNAs
to interferon .beta.(IFN.beta.) and the IFN.beta.-induced gene
RIG-I. These mRNA levels were normalized to .beta.-actin mRNA
levels and reported as relative copies of the IFN.beta. or RIG-I
mRNAs (FIG. 6A and FIG. 6B, respectively). As seen in prior
experiments, mock treatment did not induce IFN.beta. or RIG-I mRNA
expression. Similarly, GP+eVP40+2CARD-NP.sub.CT VLPs induced
IFN.beta. and RIG-I mRNA expression, as seen in prior experiments.
GP+ESAT6-eVP40 VLPs did not induce IFN.beta. and RIG-I mRNA
expression, consistent with the lack of the IFN.beta.-inducing
2CARD domain. However, GP+2CARD-ESAT6-eVP40 did induce these genes,
as expected, given the presence of 2CARD. When 2CARD-ESAT6-eVP40
was expressed without GP, the resulting VLPs did not induce
expression of IFN.beta. and RIG-I mRNA. This is consistent with the
model that GP must be present on the VLPs to allow delivery of the
2CARD-containing protein into cells to induce IFN.beta. and RIG-I
mRNA expression.
[0121] When these VLPs are used to infect a wildtype version of the
A549 cell line, the 2CARD-ESAT6-eVP40 VLPs trigger a type I
interferon (IFN) response that is as strong or stronger than the
control 2CARD-NPct+eVP40 eVLPs (which do not contain Mtb antigen)
(FIG. 7). The latter are the VLPs described above that provided
evidence for using a 2CARD-packaging eVLPs to induce a very robust
adaptive immune response. Importantly, the IFN response is
eliminated when A549 cells deleted for the cellular signaling
molecule MAVS are infected (FIG. 7). 2CARD-induced IFN responses
are expected to require MAVS as a downstream signaling molecule to
trigger IFN responses. Because the IFN response induced by
2CARD-containing eVLPs is MAVS-dependent, these results show that
the IFN induction proceeds by the expected mechanism, even in the
case of the ESAT6 containing eVLPs.
Sequence CWU 1
1
71139PRTArtificial sequenceSynthetic construct 1Thr Pro Thr Val Ala
Pro Pro Ala Pro Val Tyr Arg Asp His Ser Glu1 5 10 15Lys Lys Glu Leu
Pro Gln Asp Glu Gln Gln Asp Gln Asp His Thr Gln 20 25 30Glu Ala Arg
Asn Gln Asp Ser Asp Asn Thr Gln Ser Glu His Ser Phe 35 40 45Glu Glu
Met Tyr Arg His Ile Leu Arg Ser Gln Gly Pro Phe Asp Ala 50 55 60Val
Leu Tyr Tyr His Met Met Lys Asp Glu Pro Val Val Phe Ser Thr65 70 75
80Ser Asp Gly Lys Glu Tyr Thr Tyr Pro Asp Ser Leu Glu Glu Glu Tyr
85 90 95Pro Pro Trp Leu Thr Glu Lys Glu Ala Met Asn Glu Glu Asn Arg
Phe 100 105 110Val Thr Leu Asp Gly Gln Gln Phe Tyr Trp Pro Val Met
Asn His Lys 115 120 125Asn Lys Phe Met Ala Ile Leu Gln His His Gln
130 13528PRTArtificial sequenceSynthetic construct 2Ser Gly Gly Ser
Gly Gly Ser Gly1 53210PRTArtificial sequenceSynthetic construct
3Met Thr Thr Glu Gln Arg Arg Ser Leu Gln Ala Phe Gln Asp Tyr Ile1 5
10 15Arg Lys Thr Leu Asp Pro Thr Tyr Ile Leu Ser Tyr Met Ala Pro
Trp 20 25 30Phe Arg Glu Glu Glu Val Gln Tyr Ile Gln Ala Glu Lys Asn
Asn Lys 35 40 45Gly Pro Met Glu Ala Ala Thr Leu Phe Leu Lys Phe Leu
Leu Glu Leu 50 55 60Gln Glu Glu Gly Trp Phe Arg Gly Phe Leu Asp Ala
Leu Asp His Ala65 70 75 80Gly Tyr Ser Gly Leu Tyr Glu Ala Ile Glu
Ser Trp Asp Phe Lys Lys 85 90 95Ile Glu Lys Leu Glu Glu Tyr Arg Leu
Leu Leu Lys Arg Leu Gln Pro 100 105 110Glu Phe Lys Thr Arg Ile Ile
Pro Thr Asp Ile Ile Ser Asp Leu Ser 115 120 125Glu Cys Leu Ile Asn
Gln Glu Cys Glu Glu Ile Leu Gln Ile Cys Ser 130 135 140Thr Lys Gly
Met Met Ala Gly Ala Glu Lys Leu Val Glu Cys Leu Leu145 150 155
160Arg Ser Asp Lys Glu Asn Trp Pro Lys Thr Leu Lys Leu Ala Leu Glu
165 170 175Lys Glu Arg Asn Lys Phe Ser Glu Leu Trp Ile Val Glu Lys
Gly Ile 180 185 190Lys Asp Val Glu Thr Glu Asp Leu Glu Asp Lys Met
Glu Thr Ser Asp 195 200 205Ile Gln 2104201PRTArtificial
sequenceSynthetic construct 4Met Ser Asn Gly Tyr Ser Thr Asp Glu
Asn Phe Arg Tyr Leu Ile Ser1 5 10 15Cys Phe Arg Ala Arg Val Lys Met
Tyr Ile Gln Val Glu Pro Val Leu 20 25 30Asp Tyr Leu Thr Phe Leu Pro
Ala Glu Val Lys Glu Gln Ile Gln Arg 35 40 45Thr Val Ala Thr Ser Gly
Asn Met Gln Ala Val Glu Leu Leu Leu Ser 50 55 60Thr Leu Glu Lys Gly
Val Trp His Leu Gly Trp Thr Arg Glu Phe Val65 70 75 80Glu Ala Leu
Arg Arg Thr Gly Ser Pro Leu Ala Ala Arg Tyr Met Asn 85 90 95Pro Glu
Leu Thr Asp Leu Pro Ser Pro Ser Phe Glu Asn Ala His Asp 100 105
110Glu Tyr Leu Gln Leu Leu Asn Leu Leu Gln Pro Thr Leu Val Asp Lys
115 120 125Leu Leu Val Arg Asp Val Leu Asp Lys Cys Met Glu Glu Glu
Leu Leu 130 135 140Thr Ile Glu Asp Arg Asn Arg Ile Ala Ala Ala Glu
Asn Asn Gly Asn145 150 155 160Glu Ser Gly Val Arg Glu Leu Leu Lys
Arg Ile Val Gln Lys Glu Asn 165 170 175Trp Phe Ser Ala Phe Leu Asn
Val Leu Arg Gln Thr Gly Asn Asn Glu 180 185 190Leu Val Gln Glu Leu
Thr Gly Ser Asp 195 20059PRTArtificial sequenceSynthetic construct
5Leu Tyr Asp Arg Leu Ala Ser Thr Val1 5610PRTArtificial
sequenceSynthetic construct 6Tyr Phe Thr Phe Asp Leu Thr Ala Leu
Lys1 5 10715PRTArtificial sequenceSynthetic construct 7Pro Glu Tyr
Met Glu Ala Ile Tyr Pro Val Arg Ser Asn Ser Thr1 5 10 15
* * * * *